Microchip Zone Electrophoresis for High-Throughput Analysis of Monoclonal Antibody Charge Variants

Abstract

A high-throughput screening assay on a microfluidic chip was developed for the determination of charge variants of monocolonal antibodies (mAbs) in pI range of 7-10. This method utilizes microchip zone electrophoresis for rapid separation (<90 s) of mAb charge variants that are labeled fluorescently without altering the overall charge. The microfluidic assay achieves between 8- and 90-fold times faster separation time over conventional methods while maintaining comparable resolution and profiles of charge variant distributions. We further characterized the assay with respect to (i) the effect of pH on resolution, (ii) the effect of excipients and buffering agents, (iii) the performance of the assay compared to conventional methods, and (vi) the reproducibility of charge variant profiles. Finally, we explored the utility of the assay with four case studies: (i) monitoring C-terminal lysine modification of a mAb, (ii) quantifying the extent of deamidation of a mAb, (iii) providing charge variant information on which to base clone selection, and (iv) making process parameter-related decisions from a "design of experiment" (DoE) study. The results of these case studies demonstrate the applicability of the microfluidic assay for high-throughput monitoring of mAb quality in process development of biopharmaceuticals.

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…The use of microfluidics greatly expedites the sampling time and is conducive for use in a platform environment to screen large number of samples (Creamer, Oborny, & Lunte, 2014). In addition, the favorable mass and heat transfer in these platforms may result in good separation resolution (Han, Livingston, & Chen, 2011;Wheeler et al, 2014). Adoption of microfluidics platforms has shown promising results for these techniques, including CEX (Alekseychyk, Su, Becker, Treuheit, & Razinkov, 2017), IEF (Kinoshita, Nakatsuji, Suzuki, Hayakawa, & Kakehi, 2013), and LC-MS systems (Chambers, Mellors, Henley, & Ramsey, 2011;Dietze et al, 2016;Lotter et al, 2016;Mellors et al, 2013).…”

Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

“…A further interesting option is LIF that is already frequently used in microchip CE . A good overview about this detection mode is given by García‐Campaña et al.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Efforts are being made to develop LC columns suitable for high resolution intact protein separation [228]. At the same time, capillary zone electrophoresis (CZE) represents an alternative and complementary separation approach based on charge and hydrodynamic volume, with the potential for ultra-high efficiency [229][230][231].…”